Techniques designed to identify genes that are differentially regulated by cells under various physiological or experimental conditions (for example, differentiation, carcinogenesis, pharmacologic treatment) have become pivotal to modern biology. Differential display is one of the latest additions to the repertoire of such techniques. This technique was introduced by Liang and Pardee and described in U.S. Pat. No. 5,262,311. Prior to Liang and Pardee's introduction of this technique, those interested in identifying differentially expressed genes were compelled to resort either to differential hybridization screening (Zimmerman, G. R., et al., Cell, Vol. 21, pp. 709-715 (1980)) or to subtractive hybridization screening (St. John, T. P. et al., Cell, Vol. 16, pp. 443-452 (1979)) of complementary deoxynucleic acid ("cDNA") libraries. Neither of these methods is entirely satisfactory; both are time consuming and labor intensive. Of the two, differential hybridization (also known as .+-. screening) is particularly insensitive, being essentially confined to the detection of relatively large differences between high to moderate-abundance transcripts. Subtractive hybridization, while far more sensitive than .+-. screening, is also technically far more demanding. Furthermore, it is necessary to carry out two separate subtractive hybridization experiments in order to identify both up- and down-regulated gene expression.
Differential display offers an attractive alternative to differential and subtractive hybridization screening. Generally, Liang et al. describes a protocol which involves the reverse transcription of a messenger ribonucleic acid ("mRNA") population, in independent reactions, with each of twelve anchor primers (T.sub.12 MN), where M can be G (guanine), A (adenine) or C (cystosine) and N can be G, A, C or T (thymidine). The resulting single-stranded cDNAs are then amplified by the polymerase chain reaction (hereinafter, "PCR") using the same anchor primer used for reverse transcription together with an upstream or 5' decamer of arbitrary sequence. The PCR products, which are labeled by incorporation of tracer amounts of a radioactive nucleotide, are resolved for analysis by denaturating polyacrylamide gel electrophoresis (PAGE). This technique permits the visualization of both up- and down-regulated gene expression simultaneously in the same experiment. Liang et al. postulated that each two-primer combination could amplify only a limited subpopulation of cDNAs, and that the twelve anchor primers together with twenty arbitrary decamers (i.e., 240 PCR reactions) should result in the display of the 3' termini of all distinct mRNAs that are theoretically expressed in any given cell type (Liang, P. and Pardee, A. B., Science, Vol. 257, pp. 967-971 (1992)).
Although a number of laboratories have used Liang et al.'s differential display technique to identify differentially expressed genes, the successful implementation of differential display remains highly elusive for many researchers. Principal difficulties include poor reproducibility and large numbers of false positive results.
For example, more recently, Liang and Pardee obtained U.S. Pat. No. 5,599,672 which describes a method for isolating mRNAs as cDNAs employing a polymerase amplification method using at least two oligodeoxynucleotide primers. In one approach, the first primer contains a sequence capable of hybridizing to a site immediately upstream of the first A ribonucleotide of the mRNA's polyA tail and the second primery contains an arbitrary sequence. In another approach, the first primer contains a sequence capable of hybridizing to a site including the mRNA's poly A signal sequence and the second primer contains an arbitrary sequence. The '672 patent mentions the use of three or more nucleotides that can hybridize to an mRNA sequence that is immediately upstream of the polyA tail, however, it states that using such a first primer is not practical for rapid screening of the mRNAs contained within a given cell line due to the number of oligodeoxynucleotides required to identify every mRNA.
Villeponteau et al., in U.S. Pat. No. 5,580,726 describes a method for detecting and isolating differentially expressed mRNAs using first oligonucleotide primers for reverse transcription of mRNAs and both the first oligonucleotide primers and second oligonucleotide primers for replication of the resultant cDNAs. These primers have a length of at least twenty-one (21) nucleotides. Furthermore, Villeponteau et al. direct their claims towards the cycling parameters of the PCR reaction. Although this method may provide additional information in screening the mRNAs, as suggested by Liang and Pardee, we would expect that it would still result in a possibly significant number of false positives due to the presence of artifacts.
We have found that the problems with the prior techniques are due to several previously unrecognized and unappreciated factors that critically affect the accuracy of differential display techniques. Thus, it is an object of our invention to improve the method of using differential display techniques to identify genes.
It is another object of our invention to improve the method of using differential display techniques to identify genes to obtain better reproducibility of results.
It is yet another object of our invention to improve the method of using differential display techniques to identify genes by increasing the number of true positive results and substantially eliminating false positives.
It is another object of our invention to improve the accuracy of the results obtained by using the differential display method to identify genes.
It is yet another object of our invention to obtain pure cDNA fragments by an improved subtraction method.